Methods for assaying or isolating constituents of samples

ABSTRACT

Methods for assaying or isolating constituents of samples include applying a sample to a capillary column, which includes a matrix that includes the same material as a nonporous substrate within which the capillary column is formed, as well as drawing the sample through at least a portion of a length of the capillary column. Migration portions of the sample, such as analytes or other constituents thereof, through the capillary column may be inhibited by a stationary phase on the matrix, such as, but not limited to, capture molecules immobilized to discrete locations of the substrate. The presence of a constituent in a sample may be detected as a result of its interaction with the capillary column or a stationary phase associated with the capillary column.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/443,070,filed Nov. 18, 1999, pending, which is a divisional of application Ser.No. 09/177,814, filed Oct. 23, 1998, pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to chromatographs and other apparatus forseparating the constituents of a sample. Particularly, the presentinvention relates to a miniaturized separation apparatus which comprisesa porous capillary column. More specifically, the porous separationapparatus of the present invention includes a sample column and adetector that is disposed along the column to detect the presence of andidentify each constituent that passes by the detector. The porouscapillary column may comprise a matrix of porous silicon orhemispherical grain silicon on the surface thereof. The presentinvention also includes methods for manufacturing and using theinventive separation apparatus.

2. Background of Related Art

Various techniques have long been employed to separate the constituentsof a sample in order to facilitate the identification and quantificationof one or more of the constituents. Separation techniques are useful forseparating inorganic substances and organic substances, such aschemicals, proteins, and nucleic acids. Techniques that have beenconventionally employed for separating the constituents of a sampleinclude various types of chromatography and electrophoresis.

Chromatography is a process that is employed in analytical chemistry inorder to separate and identify the constituents of a sample. The varioustypes of chromatography that have been conventionally employed includethin layer chromatography (TLC), column chromatography, gel permeationchromatography, ion-exchange chromatography, affinity chromatography,high performance liquid chromatography (HPLC), and gas chromatography(GC).

Thin film chromatography is a well known technique wherein a drop of asample liquid is applied as a spot to a sheet of absorbent material,which may be paper or a sheet of plastic or glass covered with a thinlayer of inert absorbent material, such as cellulose or silica gel. Thinlayer chromatographic techniques typically employ a solvent mixture,such as water and an alcohol as respective stationary and mobile phases.The solvent mixture permeates the absorbent material from one edge andthe capillary action of the absorbent material moves the sample acrossthe thin layer. One of the solvents binds more tightly to the absorbentmaterial to act as a stationary phase, while the other acts as a mobilephase. As the solvent mixture moves across the absorbent material, theconstituents of the sample are separated relative to their solubility ineach of the two solvents. Stated another way, the sample constituentsequilibrate according to their relative solubilities in each of thesolvents. Constituents which are the most soluble in the stationaryphase move very little, while constituents which are more soluble in themobile phase move at higher rates and therefore travel greater distancesacross the absorbent material.

Conventional column chromatography techniques employ a vertical tube, orcolumn, that is filled with a finely divided solid, or a liquidstationary phase. As a sample is washed down through the stationaryphase, it is dissolved in and carried by a mobile phase, which istypically liquid or gas. The various constituents of the sample travelthrough the stationary phase at different rates. Thus, each of theconstituents of the sample spend a different amount of time in thecolumn. The constituents may be collected in fractions as they exit thecolumn and subsequently identified or otherwise analyzed. Constituentsof the sample which remain in the stationary phase may be separatelyidentified or otherwise analyzed by sectioning the stationary phase.

Gel permeation chromatography techniques typically employ a column witha stationary phase disposed therein. The stationary phase includes anabsorbent gel material with pores of substantially uniform size. As themobile phase and the sample that is dissolved therein pass through thestationary phase, some of the molecules that are smaller than the poresbecome entrapped therein and therefore pass through the column moreslowly. The passage of intermediately sized molecules, which are ofapproximately the same size as the pores, through the column is delayedsome, as such molecules enter some of the pores. Molecules that arelarger than the pores of the absorbent gel material pass through thestationary phase most quickly, as none of the larger molecules becomeentrapped in the pores.

Ion exchange chromatography is another variation of columnchromatography, wherein the stationary phase comprises positively ornegatively charged particles. Oppositely charged constituents of asample are attracted to the stationary phase, and therefore pass throughthe column at a slower rate than uncharged constituents and constituentswhich have the same charge as the charged particles of the stationaryphase.

In affinity chromatography, the solid phase comprises particles whichhave substrate molecules or particles, such as purified antibodies orpurified antigens, covalently attached thereto. The substrate binds to aspecific constituent or group of constituents in a sample. For example,if the stationary phase comprises antibodies that are specific for aparticular antigen, as the sample and mobile phase pass through thecolumn, only that particular antigen will be bound by the stationaryphase. The remainder of the sample constituents will pass through thecolumn quickly. The column is subsequently washed to remove any residualamount of the sample from the column. The column is then washed with adissociating solution, such as a concentrated salt solution, an acidicsolution, or a basic solution, in order to dissociate the separatedsample constituent from the stationary phase.

High performance liquid chromatography (“HPLC”) is similar to columnchromatography. In HPLC, the stationary phase is typically a liquid thatis carried on very small particles, for example 0.01 mm or less.Consequently, the stationary phase has a very large surface area, andthe mobile phase flows extremely slowly therethrough. Thus, a highpressure pump is typically employed in order to increase the rate atwhich the mobile phase moves through the column.

Conventional gas chromatography methods typically employ a liquid solidphase that is supported by a solid column and a mobile phase thatcomprises a substantially inert gas, such as nitrogen, argon, hydrogen,or helium. The sample is vaporized as it is injected into the column. Aswith thin layer chromatography, column chromatography, and HPLC, theconstituents of the sample travel across the stationary phase atdifferent rates, and therefore exit the column at different times. Asthe constituents of the sample exit the column, the constituents areanalyzed by a detector, such as a katharometer, a flame ionizer, or anelectron capture system, which generates a chromatogram. The identity ofeach constituent may then be determined by analyzing the chromatogram.

Gas chromatographs are ever-decreasing in size in order to increasetheir portability. Some small, or miniature or micro gas chromatographs,include columns, which are also referred to as capillary columns, thatare fabricated on a silicon substrate. U.S. Pat. Nos. 5,583,281 (the“'281 patent”), which issued to Conrad M. Yu on Dec. 10, 1996; 4,935,040(the “'040 patent”), which issued to Michel G. Goedert on Jun. 19, 1990;and 4,471,647 (the “'647 patent”), which issued to John H. Jerman et al.on Sep. 18, 1994, each disclose exemplary small silicon gaschromatography columns. The capillary columns that are disclosed in eachof the '281, '040, and '647 patents include open channels, or conduits,that are etched into the semiconductor substrate.

Similarly, U.S. Pat. No. 5,132,012 (the “'012 patent”), which issued toJunkichi Miura et al. on Jul. 21, 1992, discloses a liquid chromatographthat includes a capillary column formed in a semiconductor substrate.The capillary column of the chromatograph of the '012 patent comprisesan open channel, or conduit.

U.S. Pat. No. 5,571,410 (the “'410 patent”), which issued to Sally A.Swedberg et al. on Nov. 5, 1996, discloses a miniature gaschromatography system which includes a capillary column that is formedin a non-silicon substrate by laser ablation. The capillary column ofthe chromatograph of the '410 patent comprises an open channel, orconduit, with a substantially smooth surface.

The use of substantially smooth, open-channeled capillary columns inminiature chromatographs is, however, somewhat undesirable from thestandpoint that open-channeled columns typically have a surface areathat is limited by the area of the substantially smooth surface of thechannel. The amount of stationary phase material that may be disposedalong a given length of substantially smooth, open-channeled capillarycolumns is also limited by the surface area of that length of thecapillary column. Thus, in order to effectively separate the variousconstituents of a sample, the capillary column must be relatively long.Consequently, the substrate on which the capillary column is formed musthave a sufficient surface area to facilitate fabricating the capillarycolumn thereon. Thus, the use of substantially smooth, open-channeledcapillary columns in miniature gas chromatographs imposes minimum sizelimitations on such chromatographs.

Another technique for separating the various constituents of a sample istypically referred to as electrophoresis. Electrophoresis is a processwhereby molecules having a net overall electrical charge are migrated ata rate that depends on the electrical charge, size and shape of themolecule. Electrophoresis techniques typically employ a solid matrixthrough which the constituents, or molecules, of the sample aremigrated. A variation of electrophoresis that is typically referred toas polyacrylamide gel electrophoresis (PAGE) separates molecules basedstrictly on their size. In PAGE, the molecules of the sample aretypically linearized and separated, or disassociated from themselves andfrom other molecules, by means of sodium dodecyl sulfate (SDS), adetergent that binds to the hydrophobic regions of proteins, and2-mercaptoethanol, or β-mercaptoethanol, which breaks disulfide (S—S)linkages that occur between some amino acids of a protein. The sample isthen migrated through a polyacrylamide gel cross-linked matrix, whichhas very small pores. The pore size of the polyacrylamide gel may beadjusted in accordance with the molecular size, or weight, range forwhich separation is desired.

The preparation of polyacrylamide gels is a relatively long process.Moreover, the acrylamide that is used to form the gel matrix is aneurotoxin. Some of the other chemicals that may be utilized inelectrophoretic processes are also hazardous. In addition, the amount ofelectric current that may be used to separate the constituents of asample in gel electrophoresis has conventionally been limited, as toogreat a current will melt or otherwise disrupt the structure of the gel.

Thus, a small separation apparatus is needed that may be employed toconduct various types of sample separation, which is smaller thanconventional devices, and which separates samples adequately. There arealso needs for reduced equipment and operational costs.

SUMMARY OF THE INVENTION

The separation apparatus, method of manufacturing the separationapparatus, and methods of using the separation apparatus of the presentinvention address each of the foregoing needs.

The sample separation apparatus of the present invention includes asubstrate with a capillary column thereon, the latter comprising a roughsurface, such as a matrix which defines a plurality of porestherethrough or an open column with a rough surface, which is alsoreferred to as a matrix. The surface area of the matrix of eachcapillary column facilitates the separation of the constituents of asample over a relatively short length of the column compared to therequired lengths of conventional smooth, “open,” etched or ablatedcolumns to effectively separate the constituents. Preferably, thecapillary column, which is also referred to as a porous capillarycolumn, comprises porous silicon or hemispherical grain silicon, and isformed on a silicon substrate. Such a column, depending on the width anddepth thereof, may be useful for separating the constituents of a sampleor detecting constituents in a sample having a volume of as small asabout one femtoliter (1×10⁻¹⁵ liter). The separation apparatus may alsoinclude a detector disposed proximate the capillary column. Such adetector analyzes a characteristic of a constituent as the constituentpasses through the capillary column, and thereby identifies or otherwiseanalyzes the constituent.

In a first variation of the apparatus of the present invention, thesample separation apparatus may be employed as a chromatography column.Accordingly, a stationary, or solid, phase is disposed on the matrix ofthe capillary column. The type of stationary phase that is selected foruse in the sample separation apparatus is dependent upon severalfactors, including without limitation the chromatographic technique thatwill be employed with the separation apparatus and the type of sampleconstituents that are to be isolated. The types of stationary phasematerials that are useful in conventional chromatographic processes arealso useful in the first variation of the separation apparatus.

A second variation of the separation apparatus of the present inventionis useful for conducting electrophoretic separation. Thus, size of thepores that are defined through the porous silicon matrix or the amountof space between grains of hemispherical grain silicon of the capillarycolumn is determined by the desirable rate of separation and the size ofthe sample constituents for which separation is desired. The secondvariation of the separation apparatus also includes first and secondelectrodes positioned proximate respective first and second ends of thecapillary column. The first and second electrodes are connectable toopposite electrical charges so as to facilitate the generation of acurrent along a length of the capillary column, and thereby facilitatethe movement and separation of the sample constituents along the column.Preferably, the second variation of the separation apparatus alsoincludes a control column adjacent the capillary column and havingsubstantially the same dimensions, structure, and pore sizes or spacingas the capillary column. The control column is useful for determiningthe molecular size or weight of at least some of the various sampleconstituents.

In a third variation of the apparatus, the sample separation apparatusmay be employed to detect the presence or absence of increased levels ofa certain analyte. Accordingly, the third variation includes a capturesubstrate disposed on at least a portion of the rough surfaces of thecapillary column. Preferably, the capture substrate has a specificaffinity for the measured, or assayed, analyte.

A method of fabricating the sample separation apparatus of the presentinvention includes selectively forming a capillary column in asubstrate.

When a silicon substrate is employed, various techniques which are knownin the art may be employed to define a porous silicon capillary columntherein. Known techniques may also be used in order to form pores of adesired size. Known semiconductor layer formation processes may also beemployed to fabricate a detector proximate the capillary column.Similarly, known processes are useful for fabricating electrodes andother structures upon a surface of the substrate.

Capillary columns that include hemispherical grain silicon may also beselectively formed in a substrate by known techniques. First, a trench,which defines the path of the capillary column, is defined in asubstrate by known patterning processes, such as mask and etchtechniques. The surface area of the surfaces of the trench may then beincreased by known methods, such as by forming hemispherical grainsilicon thereon.

A method of utilizing the inventive separation apparatus includesdisposing a sample proximate an end of the porous capillary column anddrawing the sample through the porous capillary column to generate aflowfront of the sample and effect the separation of a constituent fromthe sample. The sample may be drawn along the capillary column bypositive pressure, negative pressure, capillary action, electriccurrent, or any other known technique that is employed to facilitate themovement of a sample along a separation apparatus.

Variations of the inventive method employ the separation apparatus ofthe present invention to effect various separation techniques,including, without limitation, various types of chromatographicseparation, electrophoresis, and the isolation and detection of one ormore analytes from a sample.

Other advantages of the present invention will become apparent to thoseof ordinary skill in the relevant art through a consideration of theappended drawings and the ensuing description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a perspective view of an embodiment of a sample separationapparatus of the present invention;

FIG. 1 a is a cross-section taken along line 1 a-1 a of FIG. 1, whichalso illustrates a sealing element disposed over at least a portion ofthe sample separation apparatus;

FIG. 1 b is a perspective view of a variation of the sample separationapparatus of FIG. 1, which illustrates an alternative placement ofdetectors;

FIG. 2 is a perspective view of a variation of the sample separationapparatus of FIG. 1 that is useful for performing chromatography;

FIG. 2 a is a perspective view of a variation of the sample separationapparatus of FIG. 2 including a vacuum source operatively connected tothe capillary column;

FIG. 3 is a perspective view of another variation of the sampleseparation apparatus of FIG. 1 that is useful for performingelectrophoresis;

FIG. 3 a is a schematic representation of the sample separationapparatus of FIG. 3, illustrating use of the sample separation apparatusin association with an electrophoresis apparatus;

FIG. 4 is a perspective view of another variation of the sampleseparation apparatus of FIG. 1 that is useful for isolating anddetecting an analyte;

FIG. 5 is a cross-sectional view of a substrate that has been patternedto define capillary column regions thereon;

FIG. 6 is an enlarged cross-sectional view taken along line 6-6 of FIG.1 and illustrating the capillary columns;

FIG. 7 is a schematic representation of the use of an anodizationchamber to porify the capillary column regions of the substrate of FIG.5; and

FIG. 8 is an enlarged cross-sectional view of an alternative roughcapillary column, which includes hemispherical grain silicon on thesurface thereof.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1, a first embodiment of a sample separationapparatus 10 of the present invention is depicted. Sample separationapparatus 10 includes a substrate 12 and capillary columns 14 formed inthe substrate. Capillary columns 14 each include a matrix 16 and aplurality of pores 18 formed through the matrix. Pores 18 permit gasesand liquids to flow along the distance of capillary columns 14.Capillary columns 14 may also include one or more reaction regions 20along the longitudinal extent thereof. Preferably, the reaction regions20 along each capillary column 14 are discrete from one another. Sampleseparation apparatus 10 may also include one or more detectors 22disposed proximate each capillary column 14.

Substrate 12 may be formed of silicon, gallium arsenide, indiumphosphide, or another material that can be treated to form porousregions, such as capillary columns 14, and upon which electricaldevices, such as detector 22, can be formed. Accordingly, capillarycolumns 14 may each comprise porous silicon.

Alternatively, capillary columns 14 may be etched into a surface ofsubstrate 12, and the surfaces of capillary columns 14 roughened. Anexemplary means of roughening the surfaces of capillary columns 14includes forming hemispherical grain silicon thereon.

FIG. 1 illustrates a sample separation apparatus 10 that includes fourcapillary columns 14. The length and porosity of each column 14 depends,in part, upon the surface tension and viscosity of the sample to bemeasured, and the desired degree of separation. As depicted, eachcapillary column 14 includes three reaction regions 20. Preferably,variations of sample separation apparatus 10 with more than onecapillary column 14 include an equal number of reaction regions 20 alongeach capillary column. Moreover, in variations of sample separationapparatus 10 wherein the capillary columns 14 each include more than onereaction region 20, the positioning and spacing between correspondingreaction regions are preferably substantially the same along each of thecapillary columns. Preferably, corresponding reaction regions 20 ondifferent columns 14 have substantially the same dimensions and pores18, or spacing between adjacent grains of hemispherical grain silicon,which spaces are also referred to as “pores,” of substantially the samesizes and porosity.

Pores 18 may have cross-sectional diameters ranging from about onenanometer (1 nm) or less to about 100 nm or greater. Due to the smallsize of pores 18, the surface tension of many liquid samples will causesuch samples to travel very slowly along the distance of capillarycolumn 14 and create a flowfront. Gaseous samples typically do notexhibit capillary action; thus, some amount of force is required tofacilitate the movement of gaseous samples along capillary column 14.Accordingly, a migration facilitator 24, such as a pump, vacuum, orcurrent-generating device, which is also referred to as a flowfacilitator, may be disposed proximate capillary column 14 in order tofacilitate or increase the migration rate of a sample 70 therealong.

Detectors 22 may be disposed adjacent capillary column 14 in order toidentify or otherwise analyze a constituent of sample 70 as theconstituent passes thereby. Various embodiments of detector 22 include,but are not limited to, thermistors, field effect transistors (FETs)that are capable of sensing various types of chemicals, components thatmeasure current as a voltage is applied to sample 70, and other devicesthat are known to measure at least one characteristic of a constituentof sample 70 or otherwise facilitate identification of the constituent.U.S. Pat. No. 5,132,012 (the “'012 patent”), which issued to JunkichiMiura et al. on Jul. 21, 1992, the disclosure of which is herebyincorporated by reference in its entirety, discloses an exemplary fieldeffect transistor that may be employed as a detector 22 in the presentinvention. U.S. Pat. No. 4,471,647 (the “'647 patent”), which issued toJohn H. Jerman et al. on Sep. 18, 1984, the disclosure of which ishereby incorporated by reference in its entirety, discloses an exemplarythermal detector that may be employed as a detector 22 in the sampleseparation apparatus of the invention. Detector 22 may be positionedproximate an exit end 14 b, which is also referred to as a second end,of capillary column 14 to analyze the various constituents of sample 70as they pass thereby. Alternatively, as shown in FIG. 1 b, detector 22may be positioned proximate a reaction region 20 of capillary column 14.More than one detector 22 may be disposed proximate each capillarycolumn 14 to analyze sample 70 and the constituents thereof at variouspositions of the capillary column.

Separation apparatus 10 may also include a processor 80 and a memorydevice 82, each of a type known in the art. Processor 80 receivesinformation about sample 70, or “sample information,” from one or moretypes of detectors 22 along column 14 and processes the sampleinformation to output same in a user-friendly format to a display 84external of sample separation apparatus 10. In processing the sampleinformation, processor 80 may compare the sample information to knowninformation that has been stored in memory device 82, and therebyidentify the sample or generate other data regarding the sampleinformation. The sample identity may then be transmitted to display 84.Following the comparison of sample information to known information,processor 80 may direct memory device 82 to store information about thesample, including its identity and associated data.

With reference to FIG. 1 a, separation apparatus 10 may also include asealing element 11 disposed over a substantial portion of the area ofeach capillary column 14 that is exposed on substrate 12. Sealingelement 11 is preferably electrically insulative and may be manufacturedfrom silicon dioxide, glass (e.g., borosilicate glass (BSG),phosphosilicate glass (PSG), borophosphosilicate glass (BPSG), etc.),silicon nitride, polyimide, other electrically non-conductive polymers,or any other electrically insulative material.

Turning now to FIG. 2, a second embodiment of the sample separationapparatus 10′ of the present invention is shown, which comprises achromatography column. Accordingly, a stationary phase 17 may bedisposed on matrix 16′ of each capillary column 14′. Stationary phase 17comprises a material that is selected on the basis of several factors,including without limitation the chromatographic technique that will beemployed and type of sample constituents for which separation orisolation is desired. Conventionally employed stationary phase materialsmay also be employed as stationary phase 17.

Separation apparatus 10′ may also include a migration facilitator 24′which comprises a pump 26′ that applies positive pressure to facilitatethe migration of a sample along each capillary column 14′. Exemplarypumps 26′ that are useful in separation apparatus 10′ are disclosed inU.S. Pat. No. 5,663,488 (the “'488 patent”), which issued to Tak KuiWang et al. on Sep. 2, 1997, the disclosure of which is herebyincorporated by reference in its entirety. Preferably, pump 26′ ispositioned proximate a sample application end 14 a′, or first end, ofeach capillary column 14′, and is in flow communication with thecapillary column and to facilitate movement of a sample 70′ along eachcolumn 14′. A valve 25′ may be disposed between pump 26′ and each column14′ in order to control the volume of gas or liquid that is forced intothe column by the pump in order to apply pressure to the column.Exemplary valves 25′ that are useful in the separation apparatus of thepresent invention include the valves that are disclosed in U.S. Pat.Nos. 4,869,282 (the “'282 patent”), which issued to Fred C. Sittler etal. on Sep. 26, 1989, and 5,583,281 (the “'281 patent”), which issued toConrad M. Yu on Dec. 10, 1996, the disclosures of each of which arehereby incorporated by reference in their entirety.

Alternatively, as depicted in FIG. 2 a, migration facilitator 24′ maycomprise a vacuum source 28′, as known in the art, which exerts anegative pressure on sample 70′ in order to pull the sample along eachcapillary column 14′. Such a vacuum source is operatively attached tocapillary column 14′, and in flow communication therewith, proximate anexit end 14 b′, or second end, thereof. Preferably, the amount ofnegative pressure that is generated by vacuum source 28′ and applied toeach capillary column 14′ may be adjusted or varied.

FIG. 3 illustrates a third embodiment of the sample separation apparatus10″ of the present invention, which is particularly useful forconducting electrophoretic separation on a sample 70″. The degree towhich the constituents of sample 70″ are separated depends upon thecross-sectional diameter of pores 18″. Accordingly, the greatest degreeof separation occurs when the size of pores 18″ is approximatelyequivalent to the size of the various constituents of sample 70″ forwhich separation is desired, or the “targeted” constituents. Thus, pores18″ of small cross-sectional diameters separate the smaller constituentsof sample 70″. Pores 18″ of larger cross-sectional diameters permit themigration and separation of the larger sized constituents through eachcapillary column 14″. Thus, the cross-sectional diameter of pores 18″preferably facilitates separation of the various targeted constituentsof sample 70″.

Electrophoretic techniques typically employ an electric current to movethe constituents of sample 70″. Thus, sample separation apparatus 10″may include a migration facilitator that comprises an electriccurrent-generating component 30. Current-generating component 30includes a first electrode 32 disposed proximate a sample applicationend 14 a″, which is also referred to as a first end, of each capillarycolumn 14″, and a second electrode 34 that is positioned proximate exitend 14 b″ of each capillary column 14″. First and second electrodes 32and 34, respectively, are fabricated from an electrically conductivematerial, and are connectable to opposite electrical charges so as tofacilitate the generation of a current along a length of the capillarycolumn. Thus, first and second electrodes 32 and 34, respectively,facilitate the migration of the constituents of sample 70″ along theirrespective capillary columns 14″ and the separation of the constituentsduring migration.

Alternatively, with reference to FIG. 3 a, a sample separation apparatus10″ which lacks a current-generating component may be utilized inassociation with a conventional electrophoresis apparatus 60 thatincludes a chamber 62 with a cathode 64 extending into one end thereofand an anode 65 extending into an opposite end of the chamber.

Referring again to FIG. 3, separation apparatus 10″ also includes acontrol column 36″ adjacent at least one of capillary columns 14″, whichhas substantially the same dimensions and a matrix 38″ and pores 40″having substantially the same configurations and sizes as the matrix 16″and pores 18″ of each capillary column 14″. Control column 36″ is usefulfor separating a control which includes markers 42 a, 42 b, 42 c, etc.of known molecular size and weight. Thus, as is known in the art, atleast some of the various constituents of the sample may be compared tomarkers 42 a, 42 b, 42 c, etc. in order to approximate the molecularsize or weight of these constituents.

Referring now to FIG. 4, a fourth embodiment of the sample separationapparatus 100 of the present invention is illustrated. Separationapparatus 100 includes a stationary phase, which is referred to ascapture substrate 117, which detects the presence and approximate levelsof a particular analyte or group of analytes in the sample. Capturesubstrate 117 may include an antibody, an antigen, or any othersubstrate material which separates a constituent from a sample on thebasis of affinity for the constituent. Accordingly, sample separationapparatus 100 comprises an assay device. Preferably, capture substrate117 has a specific affinity for the detected analyte or group ofanalytes. Capture substrate 117 is disposed along a portion of eachcapillary column 114 and securely bound to matrix 116 so as to retainsubstantially all of the capture substrate on the matrix as a samplepasses thereby. Capture substrate 117 is preferably bound to matrix 116at reaction region 120. Accordingly, detector 122 is preferablypositioned proximate reaction region 120 in order to detect whether ornot capture substrate 117 has bound an analyte.

Referring again to FIG. 1, capillary columns 14 may be formed uponsubstrate 12 by processes that are known in the art, including processesfor forming porous silicon from silicon. FIGS. 5 through 7 illustrate anexemplary process for fabricating sample separation apparatus 10. Withreference to FIG. 5, substrate 12 is appropriately patterned to definethe desired number and shapes of capillary column regions 40. As shownin FIG. 6, pores 18 are then created in the defined capillary columnregions 40, which is also referred to as “porifying” of the capillarycolumn regions, by techniques that are known in the art, such asanodization in the presence of hydrofluoric acid (HF).

Referring again to FIG. 5, patterning may include masking and etchingtechniques that are known in the art, such as those in whichphotoresists are employed. A photoresist 44 is disposed over the surfaceof substrate 12 and defined by photolithography processes, as known inthe art, to define a mask 46 with openings 48 therethrough. Openings 48expose various areas of substrate 12, which are referred to as capillarycolumn regions 40.

Patterning may also include the doping of substrate 12 with dopants andby techniques that are known in the art in order to provide the desiredamount of porosity and porous silicon of a desired morphology. As thosein the art are aware, the ability to form pores in silicon byanodization processes, as well as the size and density of such pores andthe rate at which pores are formed, depend upon the presence or absenceof dopant and the type and concentration of dopant. For example, smallpores may be formed in P−doped silicon. Larger pores are more readilyformed in P+doped silicon. N+doped silicon typically resists theformation of pores by anodization. Accordingly, patterning may alsoinclude repeated masking and differential doping of substrate 12 inorder to facilitate the subsequent selective creation of a porous matrixthrough the substrate. Such doping processes are disclosed in U.S. Pat.No. 4,532,700 (the “'700 patent”), which issued to Wayne I. Kinney etal. on Aug. 6, 1985, and U.S. Pat. No. 5,360,759 (the “'759 patent”),which issued to Reinhard Stengl et al. on Nov. 1, 1994, the disclosuresof both of which are hereby incorporated by reference in their entirety.

Alternatively, patterning may include a mask and etch, as known in theart, followed by damaging, or “roughing,” the exposed areas of substrate12 to define capillary column regions 40, as disclosed in U.S. Pat. No.5,421,958 (the “'958 patent”), which issued to Robert W. Fathauer et al.on Jun. 6, 1995, the disclosure of which is hereby incorporated byreference in its entirety. It is known in the art that porous siliconforms more readily on damaged, or roughened, areas on the surface of asilicon substrate 12. As the '958 patent discloses, the damaging ofsubstrate 12, or the creation of imperfections on same, may include,without limitation, mechanically damaging substrate 12 and applyingenergetic beams to substrate 12.

FIG. 7 schematically illustrates an anodization chamber 50 in which anexemplary process for porifying capillary column regions 40 of substrate12 (see FIG. 6) may occur. The porifying of capillary column regions 40in order to define capillary columns 14 (see FIGS. 1 and 6) in substrate12 may be performed by conventional processes, including processes forforming porous silicon regions in semiconductor devices. Exemplaryprocess for forming porous silicon from a silicon substrate aredisclosed in each of the '700, '759, and '958 patents. Such porificationprocesses typically include positioning substrate 12 within ananodization chamber 50, adjacent a partition 52, which separates theanodization chamber into a first cell 54 and a second cell 55, which arealso referred to as “sections.” An anode 56 extends into first cell 54.Similarly, a cathode 57 extends into second cell 55. Partition 52includes an opening 53 therethrough, which is covered by substrate 12and sealed to prevent the passage of liquids between first cell 54 andsecond cell 55. Thus, an upper surface 12 a of substrate 12 is exposedto first cell 54, while an opposing base surface 12 b is exposed tosecond cell 55. First cell 54 is filled with an anodizing solution 58,such as concentrated hydrofluoric acid, while second cell 55 is filledwith an electrically conductive liquid 59, such as 50% isopropylalcohol. By means of anode 56 and cathode 57, an electric current isthen applied to anodization chamber 50. As current passes throughsubstrate 12, the areas of upper surface 12 a that are exposed to firstcell 54 become porous.

The size of pores 18 is determined by, and may be varied by, varyingseveral factors, including, without limitation, the concentration of anydoped regions of the substrate, the presence or absence of dopants, thetype of dopants, the relative concentrations of the various elements ofthe anodizing solution, the duration of exposure to the anodizingsolution, the current density, the illumination, and the temperature ofthe anodizing solution.

Other known processes for patterning capillary column regions 40 onsubstrate 12 and porifying same, such as that disclosed in U.S. Pat. No.5,599,759 (the “'759 patent”), which issued to Shinji Inagaki et al. onFeb. 4, 1997, the disclosure of which is hereby incorporated byreference in its entirety, are also useful for defining capillarycolumns 14 on substrate 12, and are therefore within the scope of thefabrication process of the present invention.

With reference to FIG. 8, as another alternative, capillary columns 214that include hemispherical grain silicon 216 on the surfaces 215 thereofmay be formed in selected regions of a substrate 212 by knowntechniques. First, an elongate trench 213, which defines the path of thecapillary column, is defined in a substrate by known patterningprocesses, such as mask and etch techniques. The area of the surfaces oftrench 213 may then be increased by known methods, such as by forminghemispherical grain silicon 216 thereon. Exemplary methods of forminghemispherical grain silicon that may be employed to fabricate capillarycolumns 214 include those disclosed in U.S. Pat. No. 5,407,534, whichissued to Randhir P. S. Thakur on Apr. 18, 1995; U.S. Pat. No.5,623,243, which issued to Hirohito Watanabe et al. on Apr. 22, 1997;U.S. Pat. No. 5,634,974, which issued to Ronald A. Weimer et al. on Jun.3, 1997; U.S. Pat. No. 5,721,171, which issued to Er-Xuan Ping et al. onFeb. 24, 1998; and U.S. Pat. No. 5,726,085, which issued to DariusLammont Crenshaw et al. on Mar. 10, 1998, the disclosures of each ofwhich are hereby incorporated by reference in their entirety. Ingeneral, a film of amorphous silicon is formed in trench 213. Impuritiesare then seeded into the amorphous silicon. Then, the material isannealed to cause nucleation sites to grow at the seeding sites tothereby form the rough textured hemispherical grain silicon 216. A solidphase 218, such as a native oxide layer, may then be grown on thesurface of the hemispherical grain silicon 216. Finally, the entirestructure 210 may be enclosed by a cover layer 220 or a suitablepackage.

The hemispherical grain silicon 216 provides a rough texture on theinterior surface of the capillary column 214. The surfaces 215 ofcapillary column 214 are characterized by hemispherical ormushroom-shaped bumps, which form a porous, matrix-like structure. Thehemispherical grain silicon 216 provides at least about 1.6 to 2.2 timesthe surface area that would otherwise be provided by a conventionalsurface etched in silicon. Silicon oxide may be employed as solid phase218. Silicon oxide is a suitable solid phase material for separating ordetecting a wide variety of materials. Alternatively, materials withdifferent absorption characteristics, such as suitable resins, metals,or metal oxides, may be employed as solid phase 218.

Referring again to FIGS. 1-1 b, detector 22, processor 80, memory device82, valves 25, first electrode or cathode 32 (FIG. 3), or secondelectrode or anode 34 (FIG. 3) and other components that are carriedupon substrate 12 may be fabricated upon the substrate in a desiredlocation by known semiconductor fabrication processes. Suchsemiconductor fabrication processes include, without limitation, layerdeposition processes (e.g., sputtering and chemical vapor deposition);oxidation processes; patterning processes (e.g., masking and etching);and other conventional semiconductor device fabrication processes.

A stationary phase (see FIGS. 1 through 4) may be applied to matrix 16as known in the art.

With continued reference to FIG. 1, a method of utilizing the inventivesample separation apparatus 10 includes disposing a sample proximatefirst end 14 a of at least one capillary column 14. A liquid sample 70may then be drawn along the length of capillary columns 14 by capillaryaction or with the assistance of migration facilitator 24. A gaseoussample 70 may be drawn along the length of capillary column 14 by meansof migration facilitator 24. As sample 70 is drawn through pores 18 thatare defined by matrix 16, one or more constituents of sample 70 isseparated from the remainder of sample 70. The mechanism by which theseparation of a constituent from sample 70 occurs depends upon theseparation technique that is performed, as explained in greater detailbelow. The separated constituents may then be detected when they are inclose proximity to, or proximate, a detector 22.

Referring again to FIGS. 2 and 2 a, when sample separation apparatus 10′is employed in a chromatographic technique, one or more constituents ofa sample 70′ are separated in accordance with their relative solvenciesin stationary phase 17, which is disposed on matrix 16′, and a mobilephase, which carries the sample along the length of each capillarycolumn 14′. When either gas chromatography or HPLC is performed, the useof a pump 26′ (see FIG. 2) or a vacuum source 28′ (see FIG. 2 a) ispreferred in order to facilitate the migration of the sample along eachcapillary column 14′. Pump 26′ or vacuum source 28′ may also be employedto facilitate sample migration along capillary columns 14′ during theuse of sample separation apparatus 10′ to perform other chromatographictechniques.

Turning again to FIG. 3, in order to separate one or more constituentsof a sample 70″ by electrophoresis, the sample is first dissolved in aconventional carrier solvent, which typically includes a pH buffersolution of a desired pH, 2-mercaptoethanol, SDS, and glycerol. The SDSimparts the constituents of sample 70″ with a negative net charge andfacilitates the unraveling, or linearization, of the constituents. The2-mercaptoethanol breaks covalent disulfide (S—S) bonds between someamino acids of some protein constituents.

With continued reference to FIG. 3, a first variation of theelectrophoretic method of the present invention includes applying sample70″ to first end 14 a″ of at least one capillary column 14″. Preferably,sample 70″ is diluted in a pH-buffered solution, as known in the art. Anelectric current is then applied to current-generating component 30, inorder to migrate sample 70″ along capillary columns 14″. Preferably,first electrode 32 acts as a cathode (i.e., electrons flow therefrom),while second electrode 34 acts as an anode (i.e., electrons flowthereto).

Alternatively, with reference to FIG. 3 a, a second variation of theelectrophoretic method according to the present invention isillustrated, wherein sample separation apparatus 10″ may be disposed inan electrophoresis apparatus 60 of the type that is typically employedin gel electrophoretic techniques. Electrophoresis apparatus 60 includesa chamber 62 with a cathode 64 extending into one end thereof, and ananode 65 extending into the opposite end thereof. A buffer solution ofany of the types that are typically employed in electrophoresis, andhaving a desired pH, is poured into chamber 62. Sample separationapparatus 10″ is then positioned in electrophoresis apparatus 60, withfirst end 14 a″ of capillary columns 14″ proximate cathode 64 and secondend 14 b″ proximate anode 65. A sample 70″ is applied to first end 14a″, and an electric current of desired amperage is then applied tocathode 64 and anode 65 in order to migrate the sample along the lengthof at least one capillary column 14″.

In both the first and second variations of the electrophoretic method ofthe present invention, as the sample migrates through pores 18, theconstituents 72 a″, 72 b″, 72 c″, etc. of sample 70″ may be separated onthe basis of size or net electric charge. When separation ofconstituents 72″ on the basis of size is desired, sample 70″ preferablyincludes a substance, such as SDS, which imparts each of constituents72″ with the same net electrical charge. Various constituents of thesample may then be detected with a detector, by staining,spectrophotometrically, radiographically, or by other detection oridentification techniques that are known in the art.

As an example of the use of sample separation apparatus 100, which isillustrated in FIG. 4, a constituent, or an “analyte” 172, of a sample170 is isolated from the remainder of the sample. Sample 170 is appliedto first end 114 a of at least one capillary column 114. As sample 170moves through column 114, each of the constituents of the sample,including analyte 172, contact capture substrate 117. If sample 170includes any analytes 172 for which capture substrate 117 has anaffinity, these analytes are bound by the capture substrate 117 andisolated from the remainder of the sample as the sample contacts andpasses by the capture substrate. The presence or absence of capturesubstrate 117-bound analytes 172 may then be detected by detector 122,by staining, spectrophotometrically, radiographically, or by otherdetection or identification techniques that are known in the art. Theconcentration or relative amounts of each isolated analyte 172 may alsobe determined in such a manner.

As another example of the use of sample separation apparatus 100, todetect the presence of silver, capillary column 114 may be provided witha free chloride source, such as calcium chloride or sodium chloride.When an aqueous solution containing silver is drawn into the capillarycolumn 114, resultant precipitation of silver chloride would reduce thechloride concentration in capillary column 114. The resultant reducedionic conductivity in capillary column 114 may be measured by detector122 and compared to a conductivity profile stored in a memory elementassociated with sample separation apparatus 100. For the purpose ofcomparison, another capillary column 114′ of sample separation apparatus100 may be provided with no free chloride source. As the aqueous silversolution is drawn into the second capillary column 114′, the ionicconductivity of the second capillary column 114′ may be measured byanother detector. The ionic conductivity profile of the second capillarycolumn 114′ may be compared to that of the first capillary column 114and to the conductivity profile. The measured and stored data may thenbe processed to determine the concentration of silver in the originalsample.

Although the foregoing description contains many specifics, these shouldnot be construed as limiting the scope of the present invention, butmerely as providing illustrations of some of the presently preferredembodiments. Similarly, other embodiments of the invention may bedevised which do not depart from the spirit or scope of the presentinvention. The scope of this invention is, therefore, indicated andlimited only by the appended claims and their legal equivalents, ratherthan by the foregoing description. All additions, deletions andmodifications to the invention as disclosed herein which fall within themeaning and scope of the claims are to be embraced within their scope.

1. A method of substantially isolating a potential analyte from other components of a sample, comprising: applying a sample to a capillary column in a nonporous substrate, the capillary column comprising a matrix with at least one capture substrate immobilized relative thereto, the matrix including the same material as the nonporous substrate; and drawing the sample through at least a portion of a length of the capillary column so as to enhance separation of the constituent from the sample by the at least one capture substrate.
 2. The method of claim 1, further comprising: detecting analyte present in the sample with at least one detector disposed proximate a detecting region of the capillary column.
 3. The method of claim 1, wherein applying comprises applying a solution including the sample to the capillary column.
 4. The method of claim 1, wherein applying comprises applying the sample in a gaseous state to the capillary column.
 5. The method of claim 1, further comprising: applying a differential pressure to the capillary column to effect the drawing.
 6. The method of claim 1, wherein drawing occurs without applying differential pressure to the capillary column.
 7. The method of claim 1, wherein drawing comprises capillary action induced by the matrix.
 8. The method of claim 1, wherein drawing comprises applying an electrical current along at least a portion of a length of the capillary column.
 9. The method of claim 1, wherein applying comprises applying the sample to a capillary column including a matrix with at least one capture substrate comprising an antibody immobilized thereto.
 10. The method of claim 1, wherein applying comprises applying the sample to a capillary column including a matrix with at least one capture substrate comprising an antigen immobilized thereto.
 11. A method of identifying the presence or absence of a constituent in a sample, comprising: applying a sample to a capillary column in a nonporous substrate, the capillary column comprising a matrix including the same material as the nonporous substrate; drawing the sample through at least a portion of a length of the capillary column; and detecting a constituent present in the sample.
 12. The method of claim 11, wherein applying comprises applying a solution including the sample to the capillary column.
 13. The method of claim 11, wherein applying comprises applying the sample in a gaseous state to the capillary column.
 14. The method of claim 11, wherein drawing includes bringing the sample into contact with a stationary phase along at least a portion of the capillary column.
 15. The method of claim 14, wherein bringing the sample into contact with the stationary phase comprises bringing the sample into contact with a stationary phase disposed at a selected location along a length of the capillary column
 16. The method of claim 15, wherein detecting comprises detecting immobilization of the constituent by the stationary phase.
 17. The method of claim 16, wherein detecting comprises applying a detection reagent to at least the selected location and analyzing the detection reagent to determine whether the constituent is present.
 18. The method of claim 17, wherein analyzing comprises quantifying a change in the detection reagent.
 19. The method of claim 16, wherein detecting comprises determining an electrical characteristic of the selected location and comparing the electrical characteristic to an electrical characteristic of a control.
 20. The method of claim 15, wherein bringing the sample into contact with the stationary phase comprises bringing the sample into contact with a capture molecule that will bind to or immobilize the constituent.
 21. The method of claim 11, further comprising: applying a differential pressure to the capillary column to effect the drawing.
 22. The method of claim 11, wherein drawing occurs without applying differential pressure to the capillary column.
 23. The method of claim 22, wherein drawing comprises capillary action induced by the matrix.
 24. The method of claim 11, wherein drawing comprises applying an electrical current along at least a portion of a length of the capillary column.
 25. The method of claim 11, wherein detecting comprises evaluating the sample as it passes by a location downstream of the first end of the capillary column.
 26. The method of claim 25, wherein detecting comprises evaluating the sample as it exits the capillary column. 